Abstract
A rice cell suspension culture system with the Ramy3D promoter, which is induced by sucrose starvation, has been previously utilized to produce large quantities of recombinant proteins. Although this expression system was reported previously to generate a good yield of recombinant hGM-CSF in transgenic rice cell suspension culture, rice α-amylase was a dominant protein, with 43% of total secreted proteins and an obstacle to the production and purification of secreted recombinant proteins in a rice cell suspension culture. In this study, an intron-containing self-complementary hairpin RNA (ihpRNA)-mediated post transcriptional gene silencing (PTGS) strategy for the rice α-amylase gene was applied in order to overcome this problem in rice cell suspension culture systems. The reduction of the mRNA level of the rice α-amylase gene was verified via Northern blot analysis and siRNA, an initiator of RNA interference, was detected via an RNase protection assay. The amount of rice α-amylase in the culture medium was reduced to 8.2% as compared to that of the wild-type. A transgenic rice cell suspension culture expressing both the hGM-CSF and ihpRNA of the rice α-amylase gene demonstrated that the quantity of rice α-amylase was reduced to 22% and that the accumulation of hGM-CSF increased by 1.9-fold as compared to that in the transgenic cell line expressing hGM-CSF only. These results indicated that RNAi technology should be of great utility for suppressing undesirable genes, and should improve accumulation and facilitate the purification of secreted recombinant proteins in rice cell suspension cultures.
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References
Akazawa T, Mitsui T, Hayashi M (1988) Recent progress in α-amylase biosynthesis. In: Preiss J (ed) The biochemistry of plants: a comprehensive treatise. Academic Press, New York
Bernstein E, Caudy AA, Hammond SM et al (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366. doi:10.1038/35053110
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3
Chan MT, Yu SM (1998a) The 3′ untranslated region of a rice α-amylase gene mediates sugar-dependent abundance of mRNA. Plant J 15:685–695. doi:10.1046/j.1365-313x.1998.00244.x
Chan MT, Yu SM (1998b) The 3′ untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant. Proc Natl Acad Sci USA 95:6543–6547. doi:10.1073/pnas.95.11.6543
Chen L, Marmey P, Taylor NJ, Taylor NJ et al (1998) Expression and inheritance of multiple transgenes in rice plants. Nat Biotechnol 16:1060–1064. doi:10.1038/3455
Chu CC, Wang CC, Sun CS et al (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci Sin 18:659–668
Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in C. elegans. Nature 391:806–811. doi:10.1038/35888
Goossens A, Montafu MV, Angenon G (1999) Co-introduction of an antisense gene for an endogenous seed storage protein can increase expression of a transgene in Arabidopsis thaliana seeds. FEBS Lett 456:160–164. doi:10.1016/S0014-5793(99)00943-6
Hajdukiewicz P, Svab Z, Maliga P (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994. doi:10.1007/BF00014672
Hammond SM, Bernstein E, Beach D et al (2000) An RNA-directed nuclease mediates posttranscriptional gene silencing in Drosophila cells. Nature 404:293–296. doi:10.1038/35005107
Hirano H, Kawasaki H, Sassa H (2000) Two-dimensional gel electrophoresis using immobilized pH gradient tube gels. Electrophoresis 21:440–445. doi:10.1002/(SICI)1522-2683(20000101)21:2<440::AID-ELPS440>3.0.CO;2-X
Hong SY, Kwon TH, Jang YS et al (2006) Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures. Protein Expr Purif 47:68–73. doi:10.1016/j.pep.2005.09.028
Huang N, Sutliff TD, Litts JC et al (1990) Classification and characterization of the rice a-amylase multigene family. Plant Mol Biol 14:655–668. doi:10.1007/BF00016499
Huang J, Wu L, Yalda D et al (2002) Expression of functional recombinant human lysozyme in transgenic rice cell culture. Transgenic Res 11:229–239. doi:10.1023/A:1015663706259
Jones RL, Jacobsen JV (1991) Regulation of synthesis and transport of secreted proteins in cereal aleurone. Int Rev Cytol 126:49–88
Kusaba M, Miyahara K, Lida S et al (2003) Low glutenin content 1: a dominant mutation that suppresses the glutenin multigene family via RNA silencing in rice. Plant Cell 15:1455–1467. doi:10.1105/tpc.011452
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0
Martinez J, Patkaniowska A, Urlaub H (2002) Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563–574. doi:10.1016/S0092-8674(02)00908-X
Mitsui T, Yamaguchi J, Akazawa T (1996) Physicochemical and serological characterization of rice a-amylase isoforms and identification of their corresponding genes. Plant Physiol 110:1395–1404. doi:10.1104/pp.110.4.1395
Ogita S, Uefuji H, Yamaguchi Y et al (2003) Producing decaffeinated coffee plants. Nature 423:823. doi:10.1038/423823a
Ryu DDY, Furusaki S (1994) Advances in plant biotechnology. In: Thomas BR, Chandler J, Simmons CR et al (eds) Gene regulation and protein secretion from plant cell cultures: the rice a-amylase system. Elsevier, Amsterdam, pp 37–55
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Segal G, Song R, Messing J (2003) A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 165:387–397
Shin YJ, Hong SY, Kwon TH et al (2003) High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol Bioeng 82:778–783. doi:10.1002/bit.10635
Terashima M, Murai Y, Kawamura M et al (1999) Production of functional human a-1-antitrypsin in plant cell culture. Appl Microbiol Biotechnol 52:516–519. doi:10.1007/s002530051554
Thompson JA, Abdullah R, Cocking EC (1986) Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose. Plant Sci 47:123–133. doi:10.1016/0168-9452(86)90059-2
Wesley SV, Liu Q, Wielopolska A et al (2003) Custom knock-outs with hairpin RNA-mediated gene silencing. Methods Mol Biol 236:273–286
Whitaker JR, Sonnet PE (1989) Biocatalysis in agricultural biotechnology. In: Simmons CR, Rodriguez RL (eds) High-level synthesis and secretion of a-amylase from rice callus. ACS press, Washington, pp 202–214
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This work was supported by a grant from the Next Generation New Technology Development program of the Ministry of Commerce, Industry, and Energy and by the Post-Doc. Program of Chonbuk National University (the second half term of 2006).
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Kim, NS., Kim, TG., Jang, YS. et al. Amylase gene silencing by RNA interference improves recombinant hGM-CSF production in rice suspension culture. Plant Mol Biol 68, 369–377 (2008). https://doi.org/10.1007/s11103-008-9376-7
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DOI: https://doi.org/10.1007/s11103-008-9376-7